The long-range goal of the proposed application is characterization of nuclear transcription regulatory proteins which control osteoblast differentiation. Studies are proposed in two areas of investigation. The first three specific aims are focused on characterization of a silencer sequence in the human osteocalcin gene. The first specific aim describes studies to localize the osteocalcin silencer element (OSE) to specific nucleotides by deletion analysis, site directed mutagenesis, foot printing and mobility shift analyses. In the second specific aim, osteocalcin silencer element (OSE) homologs will be sought in other osteoblast expressed genes. In the third specific aim, OSE-binding proteins will be identified in an expression library by Southwestern screening or by protein purification and further characterized. The second area of proposed studies are embodied in specific aims 4 and 5 in which it is planned to clone and characterize osteoblast differentiation regulatory genes (ODRGs). Differential screening will be used to identify genes uniquely expressed in committed osteoblast precursors, proliferating osteoblasts, matrix producing osteoblasts or mineralization-stage osteoblasts. The genes will be characterized by partial sequence analysis and by in situ hybridization for localization in developing bones. In the 5th specific aim, functional analysis of the ODRGs will be determined by forced expression in stably transfected cell lines and by antisense oligonucleotide blocking.